Tyrosine phosphorylation and dephosphorylation of proteins is known to play a central role in regulation of cell growth and differentiation. Activation of resting T or B lymphocytes via their antigen-specific receptors (TCR and BCR, respectively) is initiated by rapid tyrosine phosphorylation of a number of regulatory proteins, such as phospholiapse Cgamma1 and phosphatidylinositol 3-kinase P13K). Identification of the protein tyrosine kinases (PTKs) involved, their regulators and their substrates, are crucial to our understanding of lymphocyte physiology in health and disease. There is considerable evidence that the src-family PTKs are important. More recent results, however, suggest that another class of non-receptor PTKs, the syk-family, also participates in the initial steps of signal transduction, and may even function upstream of the src-family PTKs. The objective of this proposal is to study the regulation of the catalytic activity of the two syk-family PTKs present in lymphoid cells, p72syk, and p70zap, and investigate their roles in the regulation of tyrosine phosphorylation in these cells. First, the properties and biology of p72syk which is not well characterized at present, will be studied by analyzing its regulation during T and B cell activation. For comparison, the related kinase p70zap will be included in the experiments. To facilitate the studies, and mapping its sites of phosphorylation, a chimeric Lyt-2/Syk molecule will be generated. This will allow direct activation of the kinase and investigations of the consequences of this activation. Second, the effects of p72syk and p70zap on three of their potential substrates, the p50csk and p56lck PTKs and P13K, will be characterized in detail. Preliminary findings indicate that p72syk may regulate the catalytic activity, binding properties and subcellular localization of these three proteins. These reactions will be analyzed in detail and the corresponding phosphorylation sites mapped and the consequences of phosphorylation studied. Overall, the proposed studies will provide a detailed characterization of the function and substrates for the syk-kinases, which seem to play an important role in lymphocyte activation perhaps proximal to the src-family kinases and other key regulatory enzymes. R43AI36028 A major goal in AIDS research is he development of new markers to monitor HIV-1 disease progression and drug therapy, particularly during the clinically latent period when its is difficult to detect HIV-1 infected cells in the peripheral blood. Recent observations using fresh lymphoid tissues have demonstrated that during this period, HIV-1 accumulates and actively replicates in lymphoid organs. Development of an imaging agent for HIV-1 disease would provide a non-invasive technique to localize and quantitative HIV-1 virions and productively infected cells in lymphatic tissues and other sites in the body. In this Phase I study, we will develop two radioiodinated CD4- immunoglobulin chimeric proteins as novel in vivo imaging agents for HIV-1 disease. The two recombinant proteins are: CD4-gamma2, a homodimer containing two chains of a CD4-human IgG2 heavy chain fusion protein; and, CD4-IgG2, a heterotetramer containing two chains of a CD4-human IgG2 heavy chain fusion protein and two chains of a CD4-human kappa light chain fusion proteins. The major advantage of CD4-based molecules over monoclonal antibodies as a candidate imaging agents is that CD4 binds with high affinity to the HIV-1 envelope glycoprotein gp120/gp41 of all strains of the virus. The development of an in vivo imaging agent for HIV-1 disease will likely provide new insights into the pathogenesis of this disease and be a valuable tool in the management of HIV-1 infected individuals.